1. Field of the Invention
The present invention includes a method for preparing stable, long-lived gas emulsions for ultrasound contrast enhancement and other uses, and to compositions of the gas emulsions so prepared. Additionally, the present invention includes precursors for preparing such emulsions.
2. Backgound of the Art
Ultrasound technology provides an important and more economical alternative to imaging techniques which use ionizing radiation. While numerous conventional imaging technologies are available, e.g., magnetic resonance imaging (MRI), computerized tomography (CT), and positron emission tomography (PET), each of these techniques use extremely expensive equipment. Moreover, CT and PET utilize ionizing radiation. Unlike these techniques, ultrasound imaging equipment is relatively inexpensive. Moreover, ultrasound imaging does not use ionizing radiation.
Ultrasound imaging makes use of differences in tissue density and composition that affect the reflection of sound waves by those tissues. Images are especially sharp where there are distinct variations in tissue density or compressibility, such as at tissue interfaces. Interfaces between solid tissues, the skeletal system, and various organs and/or tumors are readily imaged with ultrasound.
Accordingly, in many imaging applications ultrasound performs suitably without use of contrast enhancement agents; however, for other applications, such as visualization of flowing blood, there have been ongoing efforts to develop such agents to provide contrast enhancement. One particularly significant application for such contrast agents is in the area of perfusion imaging. Such ultrasound contrast agents could improve imaging of flowing blood in the heart muscle, kidneys, liver, and other tissues. This, in turn, would facilitate research, diagnosis, surgery, and therapy related to the imaged tissues. A blood pool contrast agent would also allow imaging on the basis of blood content (e.g., tumors and inflamed tissues) and would aid in the visualization of the placenta and fetus by enhancing only the maternal circulation.
A variety of ultrasound contrast enhancement agents have been proposed. The most successful have generally consisted of dispersions of small bubbles of gas that can be injected intravenously. The bubbles are injected into the bloodstream of a living body to be imaged thereby providing an emulsion in the flowing blood that is of a different density and a much higher compressibility than the surrounding fluid tissue and blood. As a result, these bubbles can easily be imaged with ultrasound.
Unfortunately, the creation of bubbles that are effective ultrasound scatterers in vivo has been difficult. Several explanations are apparent. First, such bubbles tend to shrink rapidly due to the diffusion of the trapped gas into the surrounding liquid. This is especially true of bubbles containing air or its component gases (such as nitrogen) which are highly soluble in water. It might be expected that bubble lifetime could be improved by simply increasing the size of the bubbles so more gas needs to escape before the bubbles disappear. This approach has proven unsatisfactory, however, because bubbles larger than about 10 .mu.m in diameter are cleared from the bloodstream by the lungs, preventing their further circulation. Additionally, larger bubbles are not capable of circulating through smaller blood vessels and capillaries.
Microbubbles with satisfactory in vivo performance should also posses advantageous biological characteristics. First, the compounds making up the gas inside the microbubbles should be biocompatible. Ultimately, the microbubbles containing the gas phase will decay and the gas phase will be released into the blood either as a dissolved gas or as submicron droplets of the condensed liquid. Therefore, the gases will primarily be removed from the body through lung respiration or through a combination of respiration and other metabolic pathways in the reticuloendothelial system. Even when bubble persistence is sufficient to allow for several passes through the circulatory system of an animal or human, microbubble uptake by the reticuloendothelial phagocytic cells of the liver can limit the effectiveness of the contrast agent. Adverse immune system reactions can also reduce the in vivo lifetimes of the bubble, and should be avoided. For example, "naked" microbubbles have been shown to produce adverse responses such as the activation of complement (See, for example, K. A. Shastri et al. (1991) Undersea Biomed Res., 18, 157). However, as known in the art, these undesired responses may be reduced through the use of appropriate encapsulating agents.
Accordingly, efforts to improve the in vivo lifetime, of microbubbles have included the use of stability, and hence the various encapsulating materials. For instance, gelatins or albumin microspheres that are initially formed in liquid suspension, and which entrap gas during solidification, have been used. The use of surfactants as stabilizing agents for gas bubble dispersions has also been explored, as in U.S. Pat. Nos. 4,466,442 to Hilmann et al., and 5,352,436 to Wheatley et al. Some surfactant-containing contrast enhancement agents entrap gas bubbles in the aqueous core of liposomes as in U.S. Pat. No. 5,334,381 to Unger and U.S. Pat. No. 4,900,540 to Ryan et al.
Recently, the affects of the entrapped gas on bubble lifetime has received considerable attention. Aside from air and its components, various noble gases such as krypton and argon have been used. Attention has now focused on biocompatible gases which have low water solubilities. Low solubility has been shown theoretically to be an important factor in gas bubble stability. In Epstein and Plesset, On the Stability of Gas Bubbles in Liquid-Gas Solutions, (1950) J. Chem. Phys. 18(11), 1505-1509, the rate of gas bubble shrinkage was derived as a function of gas density, solubility, and diffusivity in the surrounding medium. The stability of liquid-liquid emulsions has also been shown to increase with the decreasing solubility of the dispersed phase (Kabalnov and Shchukin, Ostwald Ripening Theory: Applications to Fluorocarbon Emulsion Stability, Advances in Colloid and Interface Science, 38:69-97, 1992).
With certain simplifying assumptions, the Epstein and Plesset formula leads to the formula for bubble lifetime (.tau.) given by Quay in U.S. Pat. No. 5,393,524: EQU .tau..alpha..rho./DC (1)
where p is the density of the entrapped gas, D is the diffusivity of the gas in the surrounding medium, and C is the solubility of the gas in the surrounding medium. Based on this formula, Quay forms bubbles using gases selected on the basis of being a gas at atmospheric pressure and body temperature (37.degree. C.) and having reduced water solubility, higher density, and reduced gas diffusivity in solution in comparison to air. In the same vein, Schneider et al. in EP0554213Al disclose gases chosen on the basis of low water solubility and high molecular weight. Specifically disclosed gases include SF.sub.6, and SeF.sub.6, as well as various perfluorinated hydrocarbons.
Although reduced water solubility and diffusivity can affect the rate at which the gas leaves the bubble (as orginally predicted by Epstein and Plesset), the Quay and Schneider gas selection criteria are inaccurate in that they result in the inclusion of certain unsuitable gases and the exclusion of certain optimally suitable gases. For example, in U.S. Pat. No. 5,393,524, Quay suggests choosing microbubble gases based on a calculation of the Q value for the proposed gas, wherein: EQU Q=4.times.10.sup.-7.times..rho./DC, (2)
.rho. is the gas density kg/m.sup.3), C is the water solubility of the gas (M), and D is the diffusivity of the gas in solution (cm.sup.2 /s). Quay teaches that the Q value should be at least 30 to be a useful gas for ultrasound contrast enhancement. A simple estimate using literature water solubility data (E. Wilhelm, R Battino, and R. J. Wilcock, Chemical Reviews, 1977, v. 77, p. 219) shows that the Q values of virtually all known gases (with the exception of hydrogen and helium) approach or exceed this value. At 25 degrees C., oxygen, for example, has a Q of 20, and nitrogen has a Q of 35. The Quay disclosure, therefore, provides little guidance for the selection of effective microbubble gases.
Moreover, the Quay Q coefficient criterion as well as Schneider's disclosure in EP0554213Al fail to consider certain major causes of bubble shrinkage, namely, the effects of bubble surface tension, surfactants and gas osmotic effects, and the potential for filling gas condensation into a liquid. Namely, the partial pressure of the filling gas must be high enough to oppose the excess Laplace overpressure inside the bubbles. If the saturated vapor pressure is low the filling gas may condense into liquid and contrast ability will be lost. Accordingly, a need exists in the art for stabilized contrast enhancement agents that are biocompatible, easily prepared, and provide superior in vivo contrast enhancement in ultrasound imaging. A need also exists for microbubble precursors and methods to prepare and use such contrast enhancement agents.